Materials employed in biomedical technology are increasingly being designed to have specific, desirable biological interactions with their surroundings, as opposed to the older, more common practice of trying to adapt traditional materials to biomedical applications. A common theme in engineering cell and tissue behavior at device surfaces is to modify the material's interface to interact selectively with a specific cell type through biomolecular recognition events. The cell surface has a variety of receptors that bind with other cells or specific proteins, which compose the environment (known as the extracellular matrix, ECM) surrounding the cells. A promising approach is the biomimetic modification of the material, in which peptides, or recombinant protein fragments containing the adhesion domains of the ECM proteins, are attached to the interface (Hubbell, Bio-tech., 1995; 13:565-576; Castner et al., Surf Sci., 2002; 500:28-60; Hartgerink et al., Proc. Nat. Acad. Sci. USA, 2002; 99:5133-5138; Tirrell et al., Surf Sci., 2002; 500:61-83; Cutler et al., Biomaterials, 2003; 24:1759-1770; Jensen et al., J. Am. Chem. Soc., 2004; 126:15223-15230; Langer et al., Nature, 2004; 428:487-492; Dankers et al., Nature Mater., 2005; 4:568-574; Semler et al., Tissue Engineering, 2005; 11:734-750; Orsello et al., Trends Biotechnol., 2001; 19:310-316).
The precise control of cell adhesion and migration in the body aids biological processes such as embryogenesis, homeostasis, the immune response, and tissue remodeling and healing (Hynes, Fibronectins, Springer Verlag, New York, 1990; Ruoslahti et al., Cell, 1994; 77). Central to this control is the integrin-mediated adhesion to proteins from the ECM. Although integrins and in particular the α5β1 integrin were originally characterized as a family of cell adhesion receptors that are responsible for anchoring cells to ECM, they have recently been shown to have a dramatic impact on dynamic processes such as mediating adenovirus infection, accelerating wound healing, providing a protection mechanism against Alzheimer's disease, and acting as a promising target for breast, colon, prostate, and rectal cancer (Vainer et al., Curr. Opin. Cell Biol., 1996; 8:724-730; Davison et al., J. Virol., 1997; 71:6204-6207; Matter et al., J. Cell Biol., 1998; 141:1019-1030; Livant et al., J. Clin. Investig., 2000; 105:1537-1545; van Golen et al., Neoplasia, 2002; 4:373-379; Kim et al., Am. J. Path., 2000; 156:1345-1362; Gong et al., Cell Growth Differ., 1997; 8:83-90; Jayne et al., EJSO, 2002; 28:30-36; Ellis, Am. Surgeon, 2003; 69:3-10; Jia et al., Cancer Res., 2004; 64:8674-8681; Chen et al., Cell Commun. Adhesion, 2004; 11:1-11).
Many therapeutic strategies require the use of peptides, such as the short sequence arginine-glycine-aspartic acid (RGD), that mimic the cell adhesion domain of fibronectin in an attempt to target the α5β1 integrin and provide treatment. Even though surface modification with a biomimetic peptide remains one of the most promising strategies, the therapeutic use of RGD-containing peptides has been limited since they cannot accurately mimic the affinity of fibronectin for the α5β1 integrin (Pierschbacher et al., Proc. Nat. Acad. Sci. USA, 1983; 80:1224-1227; Yang et al., Bone, 2001; 29:523-531; Akiyama et al., Cell Adhes. Commun., 1995; 3:13-25). This may be due to the fact that RGD peptides lack synergistic effects that come from the proline-histidine-serine-arginine-asparagine (PHSRN) site. Thus, the ability to design peptides that accurately mimic the fibronectin α5β1-mediated adhesion has increased therapeutic potential and represents a significant undertaking.
When RGD and PHSRN have been presented in a single peptide formulation in the past, results varied depending on the design. Motifs included no linker (Aucoin et al., J. Biomater. Sci. Polym. Edn., 2002; 13:447-462), or linkers of varying number of glycine (G) amino acids (G3-G13) (Kao, Biomaterials, 1999; 20:2213-2221; Kim et al., Biotech. Let., 2002; 24:2029-2033; Benoit et al., Biomaterials, 2005; 26:5209-5220), or a bivalent polyethylene glycol hybrid linker (Suzuki et al., Chem. Pharm. Bull., 2002; 50:1229-1232). However, for short periods of time before cells start secreting their own ECM, the sequences that were compared to fibronectin showed adhesion strengths that are smaller than fibronectin. Moreover, one study that examined ECM production demonstrated that ECM secretion was the lowest on surfaces functionalized with the colocalized (RGDG13PHSRN; SEQ ID NO: 11) peptide sequence compared to surfaces with a scrambled peptide sequence (RDGG13HPRNS; SEQ ID NO: 12) or RGD, emphasizing the need to design peptides that are optimized to promote cell adhesion and encourage ECM production (Benoit et al., Biomaterials, 2005; 26:5209-5220).